Positive-negative selection for homologous recombination

ABSTRACT

The invention concerns a method for the introduction of a foreign DNA into the genome of a target cell by homologous recombination as well as for the homologous recombination of suitable DNA constructs.

DESCRIPTION

The invention concerns a method for introducing a foreign DNA into thegenome of a target cell by homologous recombination as well as suitableDNA constructs for the homologous recombination.

Methods for introducing foreign DNA into the genome of eukaryotic cellsby homologous recombination are known (e.g. WO 90/11354, WO 91/09955).In this process a starting cell is transfected with a DNA constructwhich contains at least one and preferably two DNA sequence sectionsthat are homologous to regions of the genome of the cell to betransfected, a positive selection marker gene and optionally a negativeselection marker gene. In addition the DNA construct can contain aheterologous expression control sequence if it is intended to activate agene which is normally silent in the transfected cell. The transfectedcells are cultured under conditions in which a selection for thepresence of the positive selection marker gene takes place which, onexpression, leads to a selectable phenotype.

A second selection step is usually carried out in order to distinguishbetween cells in which a homologous recombination has taken place andcells in which the vector has only been randomly integrated into thegenome of the host cell. For this a negative selection marker gene isused such as the HSV thymidine kinase gene (HSV-TK) which, when present,leads to the destruction of cells in the presence of a selection agente.g. ganciclovir. In homologous recombination the cell loses the HSVthymidine kinase gene so that cells are resistant to ganciclovir. Cellsin the genome of which the targeting vector has been incorporated byrandom, non-homologous integration do not lose the HSV-TK gene and aretherefore sensitive towards ganciclovir. Cells are preferably used forthis type of selection by HSV-TK/ganciclovir which contain no functionalthymidine kinase gene (e.g. CEM tk⁻ from Ogden Bioservices Corp.,Rockville Md., USA, Cat. No. 491).

However, other host cells used for homologous recombination possesstheir own thymidine kinase gene. But this cellular thymidine kinase genecauses background problems in the negative selection. Thus for examplehomologously recombined clones may be lost during screening. Similarproblems also occur with other negative selection marker genes whichcode for a gene product whose expression must be selected against aftertransfection.

The use of polypeptides located on the cell surface as positivetransfection markers is known. Thus for example WO 95/06723 describes amethod for labelling cells using a partially deleted cell surfacereceptor gene.

In order to avoid the problems which occur with the previously usednegative selection marker genes, a negative selection marker gene isused according to the invention which codes for a polypeptide located onthe cell surface.

Hence the present invention concerns a method for introducing foreignDNA into a host cell by homologous recombination in which the host cellis transfected with a recombinant vector comprising two flankingnucleotide sequences which are homologous to a target sequence in thegenome of the host cell and inside of which a nucleotide sequence codingfor a positive selection marker is located, and a nucleotide sequenceoutside the flanking sequences which codes for a negative selectionmarker, each of the nucleotide sequences coding for the positive and thenegative selection marker being operatively linked to an expressioncontrol sequence which is active in the host cell, wherein at least onenucleotide sequence coding for a polypeptide located on the cell surfaceis used as the negative selection marker gene so that after integrationof the DNA construct into the genome of the cell by homologousrecombination the negative selection marker gene is not expressed andafter a random integration of the vector into the genome of the cell thenegative selection marker gene is expressed and its gene product ispresented on the cell surface.

Hence according to the invention a negative selection marker gene codingfor a polypeptide located on the surface is used for the homologousrecombination at an appropriate site in the vector to avoid using anegative selection method with a selection agent that is toxic for thecell. A negative selection marker gene is preferably used which codesfor a polypeptide which does not normally occur in the host cell.

Problems with toxicity or with background signals that have beendescribed for TK selection do not occur in the method according to theinvention. A further advantage of the method according to the inventionis that the number of transfected cells that have to be examined forexpression of the target gene is considerably reduced.

The host cell is preferably a eukaryotic cell, particularly preferably amammalian cell and most preferably a human cell.

In order to identify and isolate cells in which a homologousrecombination has taken place, a selection step is carried out accordingto the invention for the presence of the positive selection marker geneand a further selection step is carried out for the absence of thenegative selection marker gene.

The selection step for the presence of the positive selection markergene can be carried out in a conventional manner. Any selection markergene, and especially those suitable for eukaryotic cells, whoseexpression results in a selectable phenotype e.g. antibiotic resistanceor auxotrophy can be used as the positive selection marker gene.Antibiotic resistance genes are preferably used e.g. the neomycin,kanamycin, geneticin or hygromycin resistance gene. A particularlypreferred positive selection marker gene is the neomycinphosphotransferase gene.

The negative selection marker gene used for the method according to theinvention codes for a gene product which is presented on the surface ofthe host cell, preferably for a membrane-based polypeptide. Preferredexamples of such membrane-based polypeptides are the LNGF, the CD24, theLDL or the trk receptor or a membrane-based receptor fragment containingthe ligand binding domain of the respective receptor. Suitable receptorfragments in which the intracellular domain is completely or partiallydeleted or is modified in such a manner that the receptor presented onthe surface cannot cause signal transduction are described in WO95/06723. A particularly preferred example of such a receptor fragmentis a deletion mutant of the LNGF receptor (dLNGFR) which is a fragmentof the human low-affinity receptor of the nerve growth factor whoseintracellular and signal transducing domains have been deleted (WO95/06723).

The principle of homologous recombination under negative selection bydLNGFR is shown schematically in FIG. 1. This selection principle can ofcourse be applied to other selection marker genes coding forsurface-associated polypeptides. A plasmid is used as the recombinantvector which contains two flanking nucleic acid sections (HR1, HR2)homologous to the desired target sequence and between them the positiveselection marker gene, the neomycin resistance gene (NeoR). A nucleotidesequence coding for dLNGFR is arranged on the plasmid outside the twoflanking homologous nucleotide sequences.

The regions HR1, NeoR and HR2 are integrated into the genome when ahomologous recombination occurs with a region in the area of the targetgene (HR). However, the sequence coding for dLNGFR is not integratedinto the genome. In contrast the dLNGFR gene is retained in a formcapable of expression when the plasmid is randomly integrated into thegenome of the host cell.

The selection according to the invention for the absence of the negativeselection marker gene in the transfected host cell preferably comprisesthe steps:

(a) contacting the transfected cell with a binding molecule which bindsto the gene product of the negative selection marker gene and

(b) separating the cells containing the bound binding molecule.

Substances are used as binding molecules which can bind specifically andpreferably with high affinity to the negative selection marker.Preferably those binding molecules are used which do not have anyinterfering cross-reactivity with other surface components of the anhost cell. Examples of binding molecules are antibodies e.g. polyclonalor monoclonal antibodies, antibody fragments etc. which are directedagainst the gene product of the negative selection marker gene. Suitableantibodies to dLNGFR are for example known from WO 95/06723. When areceptor is used as a negative selection marker, a natural bindingpartner of the receptor, e.g. the receptor ligand or an analoguethereof, can of course also be used as a binding molecule. An example ofsuch a receptor ligand is NGF as a ligand of LNGFR.

In order to facilitate the separation of the cells labelled with thenegative selection marker, it is possible to use a binding moleculewhich is coupled to a solid phase and this coupling can be achieved byadsorption, covalent binding or by a high-affinity binding pair (e.g.streptavidin/biotin). The type of solid phase is generally uncriticalfor the method according to the invention and preferably those solidphases are used which enable an easy separation of the cells presentingthe negative selection marker from unlabelled cells. Therefore the solidphase can be for example present in the form of a chromatographiccolumn, but particulate solid phases such as microbeads, in particularmagnetic microbeads, which enable a particularly simple separation areespecially preferred.

Alternatively the transfected cells can also be contacted with freebinding molecules. In this case the free binding molecules preferablycarry a marker or/and a solid phase binding group. Examples for suitablemarker or/and solid phase binding groups are biotin, biotin derivatives,e.g. iminobiotin, aminobiotin or desthiobiotin, haptens, e.g.digoxigenin, fluorescein, enzymes e.g. peroxidase or alkalinephosphatase or dyes e.g. fluorescent dyes such as fluorescein,phycoerythrin, rhodamine, peridinine-chlorophyl protein, Texas red orderivatives thereof.

If a binding molecule is used which carries a solid phase binding groupsuch as biotin, a biotin derivative or a hapten, the cells labelled withthe binding molecule can be coupled to a solid phase that can react withthe solid phase binding group of the binding molecule. If a bindingmolecule is used which carries a biotin group, one can for exampleidentify the cells expressing the negative selection marker and separatethem from unlabelled cells by binding to an avidin orstreptavidin-coated solid phase.

If a binding molecule is used which carries an enzymatic marker group,the cells expressing the negative selection marker can be identifiedafter addition of an enzyme substrate by an enzyme catalysed colorreaction and optionally separated from unlabelled cells. Thisidentification can for example be carried out by putting the cells on aslide and subsequently analysing them microscopically.

If a binding molecule is used which carries a fluorescent dye, the cellsexpressing the negative selection marker can be identified by flowcytometric analysis and separated from unlabelled cells. This separationprocedure is rapid and simple and can be carried out in conventionalFACS instruments that enable the setting of fluorescence windows andcell sorting.

A further subject matter of the present invention is a recombinantvector which is suitable for use as a transfection vector in the methodaccording to the invention. This vector comprises:

(a) two flanking nucleotide sequences that are homologous to a targetsequence in a cell,

(b) a nucleotide sequence coding for a positive selection marker underthe control of an expression control sequence that is active in the celland which is located inside of the two flanking sequences according to(a),

(c) a nucleotide sequence coding for a negative selection marker underthe control of an expression control sequence that is active in the cellwhich is located outside the flanking homologous nucleotide sequencesand whose expression product is a polypeptide located on the cellsurface.

If it is intended to use the recombinant vector to activate anendogenous gene in the host cell, it contains an additional heterologousexpression control sequence which is active in the host cell between thetwo flanking homologous nucleotide sequences. This expression controlsequence comprises a promoter and preferably furtherexpression-improving sequences e.g. an enhancer. The promoter can be aregulatable or a constitutive promoter. The promoter is preferably astrong viral promoter e.g. an SV40 or a CMV promoter. The CMVpromoter/enhancer is particularly preferred.

If an amplification of the target gene in the transfected host cell isdesired, the recombinant vector contains an amplification gene betweenthe two flanking sequences. Examples of suitable amplification genes aredihydrofolate reductase, adenosine deaminase, ornithine decarboxylaseetc. A particularly preferred amplification gene is the dihydrofolatereductase gene, in particular a gene coding for a dihydrofolatereductase arginine mutant which has a lower sensitivity towards theselective agent (methotrexate) than the wild type polypeptide (Simonsenet al., Proc. Natl. Acad. Sci. USA 80 (1983), 2495).

The nucleotide sequence coding for the negative selection marker can—aselucidated above—preferably be selected from membrane-based receptors ormembrane-based receptor fragments containing the ligand binding domainof the respective receptor.

The flanking nucleotide sequences that are homologous to a targetsequence can be selected from any chromosomal regions of the genome ofthe cell to be transfected which is preferably a eukaryotic cell,particularly preferably a mammalian cell and most preferably a humancell. In the case of human cells the flanking homologous nucleotidesequences are preferably derived from the region of genes for humanfactors e.g. EPO, tPA, G-CSF, GM-CSF, TPO, interleukins, interferons,growth factors, insulin, insulin-like growth factor etc.

The flanking homologous nucleotide sequences can include the codingregion of the target gene or a part thereof. In this part they can beselected such that in a homologous recombination they cause a mutationin the coding region of the mature target polypeptide compared to theendogenous sequence present in the cell. This mutation can comprisesubstitutions, deletions and insertions of individual amino acids orwhole amino acid sections.

Yet a further subject matter of the present invention is the use ofmembrane-based surface receptors as negative selection markers in amethod of homologous recombination.

The invention is further elucidated by the following examples andfigures.

FIG. 1: shows a schematic representation of the principle of homologousrecombination using a negative selection by dLNGFR according to theinvention,

FIG. 2: shows the restriction map of the plasmid pSV-dLNGFR,

FIGS. 3a and 3 b: show results of an FACS analysis of dLNGFR-expressingand non-expressing cells,

FIG. 4: shows the restriction map of the plasmid p187-dLNGFR,

FIG. 5: shows the result of an FACS analysis to differentiate betweendLNGFR negative and positive cells.

EXAMPLES Methods Recombinant DNA Technique

Standard methods were used for the manipulation of DNA as described inSambrook, J. et al. (1989) in: Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Themolecular biological reagents were used according to the manufacturer sinstructions.

Transfection of Human Cell Lines, Cultivation and Cloning

The vector was present dissolved at a concentration of 1 μg/μl doubledistilled water. In order to ensure a high transfection efficiency, thecells were transfected with the aid of electroporation (BioRad,Genepulser™) under conditions that were-previously determined to beoptimal (960 μF/260 MV/18-22 μS). The adherently growing humanfibrosarcoma line HT1080 (ATCC CCL 121) was used as a suitable cell lineat a concentration of 10⁷ cells/0.8 ml. The cells were kept for ca. 10min on ice before and after transfection in order to reconstitute thecell membrane.

Transfected cells were sown in T-175 culture flasks and cultured in anincubator at 37° C. and 7% CO₂. After 24 h selection pressure wasapplied by adding G418 (0.8 μg/ml).

After 14 days in culture resistant clones appeared in the culture dish.After larger foci had grown, the cells were washed with PBS, trypsinizedand stained as single cell suspension.

FACS Analysis

The staining steps were carried out on ice using 10⁵ cells/preparation.The anti-dLNGFR antibody from the mouse used as the primary antibody wasdetected by adding a secondary antibody from the goat (a-mlgG-FITC,1:25, Caltag). The cells were stained with the secondary antibody aloneas a control for unspecific binding. Dead cells were detected by addingpropidium iodide (10 μg/ml). The analyses were carried out on aFACS-Vantage (Becton Dickinson Co.) according to the manufacturer'sinstructions. The specific fluorescence of cells expressing dLNGFR wasrecorded in the FL-1 channel and the dead cells in the FL-3 channel.

Example 1 Preparation of the Expression Construct for dLNGFR

The gene for dLNGFR (WO 95/06723, Boehringer Mannheim GmbH) whichcomprises 965 bp was amplified with the aid of the PCR method. Theprimers used introduced cleavage sites for the enzymes EcoRI and SalI atboth ends. After amplification the PCR fragments were cleaved with bothenzymes.

The vector pSV1 which contains the early SV40 promotor and the SV40polyA-signal (Okayama and Berg, Mol.Cell.Biol.3 (1983), 280-289; Muliganand Berg, Proc. Natl. Acad. Sci. USA 78 (1981), 2072-2076) was alsocleaved with EcoRI and SalI.

The isolated vector has a size of 3490 bp. The dLNGFR fragment isligated into the vector pSV1. The gene for dLNGFR was under theexpression control of the early SV40 promoter and the SV40 poly-signal.The entire expression cassette comprises 1900 bp. The resulting vectorPSV-DLNGR is shown in FIG. 2.

Example 2 Testing the Expression Cassette for Functionality

Cells of the line HT1080 were transiently transfected with the plasmidpSV-DLNGFR as described above. After two days growth the cells wereanalysed for expression of dLNGFR with the aid of the monoclonalanti-dLNGFR antibody. The result is shown in FIG. 3 which shows thatdLNGFR-expressing and non-expressing cells can be distinguished by FACSanalysis. It also shows that the reaction of the anti-dLNGFR antibody isspecific for transfected cells.

Example 3 Cloning the dLNGFR Expression Cassette Into a Gene TargetingVector

The dLNGFR expression cassette was isolated from pSV-DLNGFR using therestriction enzymes Notl and Pvull. The targeting vector ‘p187’ for thehuman EPO-gene (described in EP 97 112 649.5 and EP 97 112 640.5 seeFIG. 4b) was cleaved with NotI and EcoRV. The 14551 bp large vectorfragment was isolated and ligated with the dLNGFR expression cassette(FIG. 4). The resulting plasmid ‘p187-DLNGFR’ was transferred intoE.coli and propagated therein.

Example 4 Test for Negative Selection in the FACS Scan

HT1080 cells were transfected with p187-DLNGFR and selected for stableintegration i.e. G418 was added to the medium 24 hours aftertransfection. The first FACS analysis was carried out after ca. 3 weeksgrowth and namely after formation of the first foci whose cells werepooled. As shown in FIG. 5 dLNGFR negative cells, in this case 14% ofthe population, can at this time be distinguished from thedLNGFR-expressing cells by FACS analysis.

In addition to the rarely occurring event of homologous recombinationthis cell population, also contains cells whose surface receptor densityis too low and therefore are not recognized by the detection system.However, in this manner the number of clones which have to besubsequently tested for the expression of the target gene can beconsiderably reduced (in this case 14 of 100%).

If in a transfection preparation no clone is present which contains ahomologously recombined targeting vector, then this situation can beindicated with much less work compared to the conventional screening.The absence of homologously recombined clones is demonstrated by theoccurrence of a population reacting 100% with anti-dLNGFR antibodies. Inthis case it is not necessary to screen further for the expression oftarget gene.

What is claimed is:
 1. A method for introducing foreign DNA into a hostcell by homologous recombination, comprising transfecting the host cellwith a recombinant vector comprising: a) two flanking nucleotidesequences which are homologous to a target gene sequence in the genomeof the host cell, b) a nucleotide sequence encoding a positive selectionmarker located between the two flanking nucleotide sequences, c)nucleotide sequence encoding a negative selection marker located outsidethe two flanking sequences, and d) each of b) and c) are operativelylinked to an expression control sequence which is active in the hostcell, wherein the negative selection marker comprises at least onenucleotide sequence encoding a polypeptide located on the cell surface,and wherein the negative selection marker is not expressed after atargeted integration of the vector into the genome of the cell byhomologous recombination, and is expressed and presented on the cellsurface after a random integration of the vector into the genome of thecell.
 2. The method as claimed in claim 1, wherein a selection stepcombines detecting the expression of the positive selection marker andthe negative selection marker.
 3. The method as claimed in claim 2,wherein detecting the expression of the negative selection markercomprises the steps of: (a) contacting a transfected cell with a bindingmolecule which binds to the polypeptide located on the cell surface; and(b) identifying the transfected cell having a bound binding molecule,and wherein the cells containing the bound binding molecule areseparated from cells without a bound binding molecule.
 4. The method asclaimed in claim 3, wherein the binding molecule is an antibody.
 5. Themethod as claimed in claim 3, wherein the binding molecule is a ligand.6. The method as claimed in claim 3, wherein the binding molecule iscoupled to a solid phase.
 7. The method as claimed in claim 6, whereinthe solid phase is a magnetic microbead.
 8. The method as claimed inclaim 3, wherein the binding molecule is conjugated to a marker and/or asolid phase binding group.
 9. The method as claimed in claim 8, whereinthe marker and/or the solid phase binding group is selected from thegroup consisting of a biotin molecule, a biotin derivative, a hapten, anenzyme and a dye.
 10. The method as claimed in claim 9, wherein themarker, solid phase binding group, or a combination thereof, is selectedfrom the group consisting of biotin, iminobiotin, aminobiotin anddesthiobiotin.
 11. The method as claimed in claim 10, wherein the biotinmolecule or the biotin derivative is detected by binding to an avidin ora streptavidin-coated solid phase.
 12. The method as claimed in claim 9,wherein the enzyme is an alkaline phosphatase or a peroxidase.
 13. Themethod as claimed in claim 12, wherein the enzyme is identified by anenzyme-catalyzed color reaction.
 14. The method as claimed in claim 9,wherein the dye is a fluorescent dye.
 15. The method as claimed in claim14, wherein the fluorescent dye is fluorescein, phycoerythrin,rhodamine, peridininechlorophyl protein or Texas red.
 16. The method asclaimed in claim 14, comprising detecting the dye or the fluorescent dyeby flow-cytometric analysis.
 17. The method as claimed in claim 1,wherein the cell is a eukaryotic cell.
 18. The method according to claim17, wherein the cell is a mammalian cell.
 19. The method according toclaim 17 or 18, wherein the cell is a human cell.
 20. The method asclaimed in claim 1, wherein the nucleotide sequence for the negativeselection marker encodes a receptor wherein the receptor is at least oneof an LNGF receptor, a CD24 receptor, an LDL receptor, a trk receptor,and a membrane-based receptor or a fragment thereof, and wherein saidreceptor or fragment thereof encoded by the negative selection markercomprises a domain for binding extracellular ligands.
 21. A recombinantvector comprising a) two flanking nucleotide sequences that arehomologous to a target gene sequence in a cell, b) a nucleotide sequenceencoding a positive selection marker under the control of an expressioncontrol sequence that is active in the cell, wherein the nucleotidesequence is located inside of the two flanking sequences according to(a), and c) a nucleotide sequence encoding a negative selection markerunder the control of an expression control sequence that is active inthe cell, wherein the nucleotide sequence of c) is located outside theflanking homologous nucleotide sequences according to (a), and anexpression product of the nucleotide is a polypeptide located on thecell surface.
 22. The vector as claimed in claim 21, wherein the twoflanking nucleotide sequences which contain the target gene sequence areselected from the group consisting of EPO, tPA, G-CSF, GM-CSF,thrombopoietin, an interleukin, an interferon, a growth factor, insulinor insulin-like growth factor.
 23. The vector as claimed in claim 21,wherein the nucleotide sequence encoding the positive selection markeris a drug-resistance gene selected from the group consisting ofneomycin, kanamycin, geneticin and hygromycin.
 24. The vector as claimedin claim 21, wherein a further expression control sequence is locatedinside of the flanking sequences.
 25. The vector as claimed in claim 24,wherein the expression control sequence comprises a CMV promoter. 26.The vector as claimed in claim 21, further comprising an amplificationsequence positioned inside of the flanking sequences.
 27. The vector asclaimed in claim 21, wherein the two flanking nucleotide sequencescontain the coding region of the target gene or a part thereof.
 28. Thevector as claimed in claim 27, wherein the two flanking nucleotidesequences are sequences for introducing a mutation within the codingregion of the nucleotide sequence encoding the mature target polypeptideby homologous recombination.
 29. The vector as claimed in claim 21,wherein the nucleotide sequence encoding the negative selection markerencodes a membrane-based molecule selected from the group consisting ofa LNGF receptor, a CD24 receptor, a LDL receptor, a trk receptor, and amembrane-based receptor or a fragment thereof, and wherein themembrane-based molecule encoded by the negative selection markercomprises a domain for binding extracellular ligands.
 30. A recombinantvector comprising a) two flanking nucleotide sequences that arehomologous to a target gene sequence in a cell, b) a nucleotide sequenceencoding a positive selection marker under the control of an expressioncontrol sequence that is active in the cell, wherein the nucleotidesequence is located inside of the two flanking sequences according to(a), and c) a nucleotide sequence encoding a negative selection markerunder the control of an expression control sequence that is active inthe cell, wherein the nucleotide sequence of c) is located outside theflanking homologous nucleotide sequences according to (a), and anexpression product of the nucleotide sequence is a receptor fragmentcontaining an extracellular ligand binding domain and located on thecell surface, with the proviso that an intracellular domain of thereceptor fragment is fully or partially deleted or modified, and whereinby deleting or modifying said intracellular domain of said receptorfragment, said receptor fragment is incapable of signal transduction.31. A method for endogenous gene activation comprising introducingforeign DNA into a host cell by homologous recombination, comprisingtransfecting the host cell with a recombinant vector comprising: a) twoflanking nucleotide sequences which are homologous to a target genesequence in the genome of the host cell, b) a nucleotide sequenceencoding a positive selection marker located between the two flankingnucleotide sequences, c) a nucleotide sequence encoding a negativeselection marker located outside the two flanking sequences, and d) eachof b) and c) are operatively linked to a heterologous expression controlsequence which is active in the host cell, and e) an additionalheterologous expression control sequence which is active in the hostcell, and which is between the two flanking homologous nucleotidesequences, wherein the negative selection marker comprises at least onenucleotide sequence encoding a polypeptide located on the cell surface,and wherein the negative selection marker is not expressed after atargeted integration of the vector into the genome of the cell byhomologous recombination and is expressed and presented on the cellsurface after a random integration of the vector into the genome of thecell.
 32. The method according to claim 31, wherein the heterologousexpression control sequence of e) is a regulatable promoter or aconstitutive promoter.